Biosynthetic Conclusions from the Functional Dissection of Oxygenases for Biosynthesis of Actinorhodin and Related Streptomyces Antibiotics

Actinorhodin (ACT) produced by Streptomyces coelicolor A3(2) belongs to the benzoisochromanequinone (BIQ) class of antibiotics. ActVA-ORF5, a flavin-dependent monooxygenase (FMO) essential for ACT biosynthesis, forms a two-component enzyme system in combination with a flavin:NADH oxidoreductase, ActVB. The genes for homologous two-component FMOs are found in the biosynthetic gene clusters for two other BIQs, granaticin (GRA) and medermycin (MED), and a closely related antibiotic, alnumycin (ALN). Our functional analysis of these FMOs (ActVA-ORF5, Gra-ORF21, Med-ORF7, and AlnT) in S. coelicolor unambiguously demonstrated that ActVA-ORF5 and Gra-ORF21 are bifunctional and capable of both p-quinone formation at C-6 in the central ring and C-8 hydroxylation in the lateral ring, whereas Med-ORF7 catalyzes only p-quinone formation. No p-quinone formation on a BIQ substrate was observed for AlnT, which is involved in lateral p-quinone formation in ALN. [Display omitted] ► ActVA-ORF5 is a flavin-dependent monooxygenase required for actinorhodin biosynthesis ► ActVA-ORF5 and its three close homologs were functionally dissected ► ActVA-ORF5 and Gra-21 are bifunctional at C-6/C-8, while Med-7 acts only for C-6 ► AlnT exhibits different regiospecificity for oxidation of tricyclic substrates Actinorhodin (ACT) belongs to the benzoisochromanequinone class of antibiotics. Taguchi et al. show that ActVA-ORF5 and Gra-ORF21, flavin-dependent monooxygenases involved in biosynthesis of ACT and a related antibiotic, catalyze oxygenations at two different carbons in the same substrate, which is rarely seen.